Estrogen receptor beta

ABSTRACT

The present invention relates to isolated DNA encoding novel estrogen receptors, the proteins encoded by said DNA, chimeric receptors comprising parts of said novel receptors and uses thereof.

This application is a divisional of application Ser. No. 08/826,361,filed Mar. 26, 1997.

This invention relates to the field of receptors belonging to thesuperfamily of nuclear hormone receptors, in particular to steroidreceptors. The invention relates to DNA encoding a novel steroidreceptor, the preparation of said receptor, the receptor protein, andthe uses thereof.

Steroid hormone receptors belong to a superfamily of nuclear hormonereceptors involved in ligand-dependent transcriptional control of geneexpression. In addition, this superfamily consists of receptors fornon-steroid hormones such as vitamine D, thyroid hormones and retinoids(Giguère et al, Nature 330, 624-629, 1987; Evans, R. M., Science 240,889-895,1988). Moreover, a range of nuclear receptor-like sequences havebeen identified which encode socalled ‘orphan’ receptors: thesereceptors are structurally related to and therefore classified asnuclear receptors, although no putative ligands have been identified yet(B. W. O'Malley, Endocrinology 125, 1119-1170, 1989; D. J. Mangelsdorfand R. M. Evans, Cell, 83, 841-850, 1995).

The superfamily of nuclear hormone receptors share a modular structurein which six distinct structural and functional domains, A to F, aredisplayed (Evans, Science 240, 889-895, 1988). A nuclear hormonereceptor is characterized by a variabel N-terminal region (domain A/B),followed by a centrally located, highly conserved DNA-binding domain(hereinafter referred to as DBD; domain C), a variable hinge region(domain D), a conserved ligand-binding domain (herein after referred toas LBD; domain E) and a variable C-terminal region (domain F).

The N-terminal region, which is highly variable in size and sequence, ispoorly conserved among the different members of the superfamily. Thispart of the receptor is involved in the modulation of transcriptionactivation (Bocquel et al, Nucl. Acid Res., 17, 2581-2595, 1989; Tora etal, Cell 59, 477-487, 1989).

The DBD consists of approximately 66 to 70 amino acids and isresponsible for DNA-binding activity: it targets the receptor tospecific DNA sequences called hormone responsive elements (hereinafterreferred to as HRE) within the transcription control unit of specifictarget genes on the chromatin (Martinez and Wahli, In ‘Nuclear HormoneReceptors’, Acad. Press, 125-153, 1991).

The LBD is located in the C-terminal part of the receptor and isprimarily responsible for ligand binding activity. In this way, the LBDis essential for recognition and binding of the hormone ligand and, inaddition possesses a transcription activation function, therebydetermining the specificity and selectivity of the hormone response ofthe receptor. Although moderately conserved in structure, the LBD's areknown to vary considerably in homology between the individual members ofthe nuclear hormone receptor superfamily (Evans, Science 240, 889-895,1988; P. J. Fuller, FASEB J., 5, 3092-3099, 1991; Mangelsdorf et al,Cell, Vol. 83, 835-839, 1995).

Functions present in the N-terminal region, LBD and DBD operateindependently from each other and it has been shown that these domainscan be exchanged between nuclear receptors (Green et al, Nature, Vol.325, 75-78, 1987). This results in chimeric nuclear receptors, such asdescribed for instance in WO-A-8905355.

When a hormone ligand for a nuclear receptor enters the cell bydiffusion and is recognized by the LBD, it will bind to the specificreceptor protein, thereby initiating an allosteric alteration of thereceptor protein. As a result of this alteration the ligand/receptorcomplex switches to a transcriptionally active state and as such is ableto bind through the presence of the DBD with high affinity to thecorresponding HRE on the chromatin DNA (Martinez and Wahli, ‘NuclearHormone Receptors’, 125-153, Acad. Press, 1991). In this way theligand/receptor complex modulates expression of the specific targetgenes. The diversity achieved by this family of receptors results fromtheir ability to respond to different ligands.

The steroid hormone receptors are a distinct class of the nuclearreceptor superfamily, characterized in that the ligands are steroidhormones. The receptors for glucocorticoids (GR), mineralcorticoids(MR), progestins (PR), androgens (AR) and estrogens (ER) are classicalsteroid receptors. Furthermore, the steroid receptors have the uniqueability upon activation to bind to palindromic DNA sequences, theso-called HRE's, as homodimers. The GR, MR, PR and AR recognize the sameDNA sequence, while the ER recognizes a different DNA sequence. (Beatoet al, Cell, Vol. 83, 851-857, 1995). After binding to DNA, the steroidreceptor is thought to interact with components of the basaltranscriptional machinery and with sequence-specific transcriptionfactors, thus modulating the expression of specific target genes.

Several HRE's have been identified, which are responsive to thehormone/receptor complex. These HRE's are situated in thetranscriptional control units of the various target genes such asmammalian growth hormone genes (responsive to glucocorticoid, estrogen,testosterone), mammalian prolactin genes and progesterone receptor genes(responsive to Estrogen), avian ovalbumin genes (responsive toprogesterone), mammalian metallothionein gene (responsive toglucocorticoid) and mammalian hepatic α_(2μ)-globulin gene (responsiveto estrogen, testosterone, glucocorticoid).

The steroid hormone receptors have been known to be involved inembryonic development, adult homeostasis as well as organ physiology.Various diseases and abnormalities have been ascribed to a disturbancein the steroid hormone pathway. Since the steroid receptors exercisetheir influence as hormone-activated transcriptional modulators, it canbe anticipated that mutations and defects in these receptors, as well asoverstimulation or blocking of these receptors might be the underlyingreason for the altered pattern. A better knowledge of these receptors,their mechanism of action and of the ligands which bind to said receptormight help to create a better insight in the underlying mechanism of thehormone signal transduction pathway, which eventually will lead tobetter treatment of the diseases and abnormalities linked to alteredhormone/receptor functioning.

For this reason cDNA's of the steroid and several other nuclearreceptors of several mammalians, including humans, have been isolatedand the corresponding amino acid sequences have been deduced, such asfor example the human steroid receptors PR, ER, GR, MR, and AR, thehuman non-steroid receptors for vitamine D, thyroid hormones, andretinoids such as retinol A and retinoic acid. In addition, cDNA'sencoding well over 100 mammalian orphan receptors have been isolated,for which no putative ligands are known yet (Mangelsdorf et al, Cell,Vol.83, 835-839, 1995). However, there is still a great need for theelucidation of other nuclear receptors, in order to unravel the variousroles these receptors play in normal physiology and pathology.

The present invention provides for such a novel nuclear receptor. Morespecific, the present invention provides for novel steroid receptors,having estrogen mediated activity. Said novel steroid receptors arenovel estrogen receptors, which are able to bind and be activated by,for example, estradiol, estrone and estriol.

According to the present invention it has been found that a novelestrogen receptor is expressed as an 8 kb transcript in human thymus,spleen, peripheral blood lymphocytes (PBLs), ovary and testis.Furthermore, additional transcripts have been identified. Anothertranscript of approximately 10 kb was identified in ovary, thymus andspleen. In testis, an additional transcript of 1.3 kb was detected.These transcripts are probably generated by alternative splicing of thegene encoding the novel estrogen receptor according to the invention.

Cloning of the cDNA's encoding the novel estrogen receptors according tothe invention revealed that several splicing variants of said receptorcan be distinguished. At the protein level, these variants differ onlyat the C-terminal part.

cDNA encoding an ER has been isolated (Green, et al, Nature 320,134-139, 1986; Greene et al, Science 231, 1150-1154, 1986), and thecorresponding amino acid sequence has been deduced. This receptor andthe receptor according to the present invention, however, are distinct,and encoded for by different genes with different nucleic acidsequences. Not only do the ER of the prior art (hereinafter referred toas classical ER) and the ER according to the present invention differ inamino acid sequence, they also are located on different chromosomes. Thegene encoding the classical ER is located on chromosome 6, whereas thegene encoding the ER according to the invention was found to be locatedon chromosome 14. The ER according to the invention furthermoredistinguishes itself from the classical receptor in differences intissue distribution, indicating that there may be important differencesbetween these receptors at the level of estrogenic signalling.

In addition, two orphan receptors, ERRα and ERRβ, having an estrogenreceptor related structure have been described (Giguère et al, Nature331, 91-94, 1988). These orphan receptors, however, have not beenreported to be able to bind estrodial or any other hormone that binds tothe classical ER, and other ligands which bind to these receptors havenot been found yet. The novel estrogen receptor according to theinvention distinguishes itself clearly from these receptors since it wasfound to bind estrogens.

The fact that a novel ER according to the invention has been found isall the more surprising, since any suggestion towards the existence ofadditional estrogen receptors was absent in the scientific literature:neither the isolation of the classical ER nor the orphan receptors ERRαand ERRβ suggested or hinted towards the presence of additional estrogenreceptors such as the receptors according to the invention. Theidentification of additional ER's could be a major step forward for theexisting clinical therapies, which are based on the existence of one ERand as such ascribe all estrogen mediated abnormalities and/or diseasesto this one receptor. The receptors according to the invention will beuseful in the development of hormone analogs that selectively activateeither the classical ER or the novel estrogen receptor according to theinvention. This should be considered as one of the major advantages ofthe present invention.

Thus, in one aspect, the present invention provides for isolated cDNAencoding a novel steroid receptor. In particular, the present inventionprovides for isolated cDNA encoding a novel estrogen receptor.

According to this aspect of the present invention, there is provided anisolated DNA encoding a steroid receptor protein having an N-terminaldomain, a DNA-binding domain and a ligand-binding domain, wherein theamino acid sequence of said DNA-binding domain of said receptor proteinexhibits at least 80% homology with the amino acid sequence shown in SEQID NO:3, and the amino acid sequence of said ligand-binding domain ofsaid receptor protein exhibits at least 70% homology with the amino acidsequence shown in SEQ ID NO:4.

In particular, the isolated DNA encodes a steroid receptor proteinhaving an N-terminal domain, a DNA-binding domain and a ligand-bindingdomain, wherein the amino acid sequence of said DNA-binding domain ofsaid receptor protein exhibits at least 90%, preferably 95%, morepreferably 98%, most preferably 100% homology with the amino acidsequence shown in SEQ ID NO:3.

More particularly, the isolated DNA encodes a steroid receptor proteinhaving an N-terminal domain, a DNA-binding domain and a ligand-bindingdomain , wherein the amino acid sequence of said ligand-binding domainof said receptor protein exhibits at least 75%, preferably 80%, morepreferably 90%, most preferably 100% homology with the amino acidsequence shown in SEQ ID NO:4.

A preferred isolated DNA according to the invention encodes a steroidreceptor protein having the amino acid sequence shown in SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:21 or SEQ ID NO:25.

A more preferred isolated DNA according to the invention is an isolatedDNA comprising a nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:20 or SEQ ID NO:24.

The DNA according to the invention may be obtained from cDNA.Alternatively, the coding sequence might be genomic DNA, or preparedusing DNA synthesis techniques.

The DNA according to the invention will be very useful for in vivoexpression of the novel receptor proteins according to the invention insufficient quantities and in substantially pure form.

In another aspect of the invention, there is provided for a steroidreceptor comprising the amino acid sequence encoded by the abovedescribed DNA molecules.

The steroid receptor according to the invention has an N-terminaldomain, a DNA-binding domain and a ligand-binding domain, wherein theamino acid sequence of said DNA-binding domain of said receptor exhibitsat least 80% homology with the amino acid sequence shown in SEQ ID NO:3,and the amino acid sequence of said ligand-binding domain of saidreceptor exhibits at least 70% homology with the amino acid sequenceshown in SEQ ID NO:4.

In particular, the steroid receptor according to the invention has anN-terminal domain, a DNA-binding domain and a ligand-binding domain,wherein the amino acid sequence of said DNA-binding domain of saidreceptor exhibits at least 90%, preferably 95%, more preferably 98%,most preferably 100% homology with the amino acid sequence shown in SEQID NO:3.

More particular, the steroid receptor according to the invention has anN-terminal domain, a DNA-binding domain and a ligand-binding domain,wherein the amino acid sequence of said ligand-binding domain of saidreceptor exhibits at least 75%, prefearbly 80%, more preferably 90%,most preferably 100% homology with the amino acid sequence shown in SEQID NO:4.

It will be clear for those skilled in the art that also steroid receptorproteins comprising combined DBD and LBD preferences and DNA encodingsuch receptors are subject of the invention.

Preferably, the steroid receptor according to the invention comprises anamino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:21 orSEQ ID NO:25.

Also within the scope of the present invention are steroid receptorproteins which comprise variations in the amino acid sequence of the DBDand LBD without loosing their respective DNA-binding or ligand-bindingactivities. The variations that can occur in those amino acid sequencescomprise deletions, substitutions, insertions, inversions or additionsof (an) amino acid(s) in said sequence, said variations resulting inamino acid difference(s) in the overall sequence. It is well known inthe art of proteins and peptides that these amino acid differences leadto amino acid sequences that are different from, but still homologouswith the native amino acid sequence they have been derived from.

Amino acid substitutions that are expected not to essentially alterbiological and immunological activities, have been described in forexample Dayhof, M. D., Atlas of protein sequence and structure, Nat.Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3. Amino acidreplacements between related amino acids or replacements which haveoccurred frequently in evolution are, inter alia Ser/Ala, Ser/Gly,Asp/Gly, Arg/Lys, Asp/Asn, Ile/Val. Based on this information Lipman andPearson developed a method for rapid and sensitive protein comparison(Science 227, 1435-1441, 1985) and determining the functional similaritybetween homologous polypeptides.

Variations in amino acid sequence of the DBD according to the inventionresulting in an amino acid sequence that has at least 80% homology withthe sequence of SEQ ID NO:3 will lead to receptors still havingsufficient DNA binding activity. Variations in amino acid sequence ofthe LBD according to the invention resulting in an amino acid sequencethat has at least 70% homology with the sequence of SEQ ID NO:4 willlead to receptors still having sufficient ligand binding activity.

Homology as defined herein is expressed in percentages, determined viaPCGENE. Homology is calculated as the percentage of identical residuesin an alignment with the sequence according to the invention. Gaps areallowed to obtain maximum alignment.

Comparing the amino acid sequences of the classical ER and the ER'saccording to the invention revealed a high degree of similarity withintheir respective DBD's. The conservation of the P-box (amino acidsE-G-X-X-A) which is responsible for the actual interactions of theclassical ER with the target DNA element (Zilliacus et al., Mol.Endo. 9,389, 1995; Glass, End.Rev. 15, 391, 1994), is indicative for arecognition of estrogen responsive elements (ERE's) by the ER'saccording to the invention. The receptors according to the inventionindeed showed ligand-dependent transactivation on ERE-containingreporter constructs. Therefore, the classical ER and the novel ER'saccording to the invention may have overlapping target genespecificities. This could indicate that in tissues which co-express bothrespective ER's, these receptors compete for ERE's. The ER's accordingto the invention may regulate transcription of target genes differentlyfrom classical ER regulation or could simply block classical ERfunctioning by occupying estrogen responsive elements. Alternatively,transcription might be influenced by heterodimerization of the differentreceptors.

Thus, a preferred steroid receptor according to the invention comprisesthe amino acid sequence E-G-X-X-A within the P box of the DNA bindingdomain, wherein X stands for any amino acid. Also within the scope ofthe invention is isolated DNA encoding such a receptor.

Methods to prepare the receptors according to the invention are wellknown in the art (Sambrook et al., Molecular Cloning: a LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989).The most practical approach is to produce these receptors by expressionof the DNA encoding the desired protein.

A wide variety of host cell and cloning vehicle combinations may beusefully employed in cloning the nucleic acid sequence coding for thereceptor of the invention. For example, useful cloning vehicles mayinclude chromosomal, non-chromosomal and synthetic DNA sequences such asvarious known bacterial plasmids and wider host range plasmids andvectors derived from combinations of plasmids and phage or virus DNA.Useful hosts may include bacterial hosts, yeasts and other fungi, plantor animal hosts, such as Chinese Hamster Ovary (CHO) cells or monkeycells and other hosts.

Vehicles for use in expression of the ligand-binding domain of thepresent invention will further comprise control sequences operablylinked to the nucleic acid sequence coding for the ligand-bindingdomain. Such control sequences generally comprise a promoter sequenceand sequences which regulate and/or enhance expression levels.Furthermore an origin of replication and/or a dominant selection markerare often present in such vehicles. Of course control and othersequences can vary depending on the host cell selected.

Techniques for transforming or transfecting host cells are quite knownin the art (see, for instance, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, 1989).

Recombinant expression vectors comprising the DNA of the invention aswell as cells transformed with said DNA or said expression vector alsoform part of the present invention.

In a further aspect of the invention, there is provided for a chimericreceptor protein having an N-terminal domain, a DNA-binding domain, anda ligand-binding domain, characterized in that at least one of thedomains originates from a receptor protein according to the invention,and at least one of the other domains of said chimeric proteinoriginates from another receptor protein from the nuclear receptorsuperfamily, provided that the DNA-binding domain and the ligand-bindingdomain of said chimeric receptor protein originate from differentproteins.

In particular, the chimeric receptor according to the inventioncomprises the LBD according to the invention, said LBD having an aminoacid sequence which exhibits at least 70% homology with the amino acidsequence shown in SEQ ID NO:4. In that case the N-terminal domain andDBD should be derived from another nuclear receptor, such as for examplePR. In this way a chimeric receptor is constructed which is activated bya ligand of the ER according to the invention and which targets a geneunder control of a progesterone responsive element. The chimericreceptors having a LBD according to the invention are useful for thescreening of compounds to identify novel ligands or hormone analogswhich are able to activate an ER according to the invention.

In addition, chimeric receptors comprising a DBD according to theinvention, said DBD having an amino acid sequence exhibiting at least80% homology with the amino acid sequence shown in SEQ ID NO:3, and aLBD and, optionally, an N-terminal domain derived from another nuclearreceptor, can be succesfully used to identify novel ligands or hormoneanalogs for said nuclear receptors. Such chimeric receptors areespecially useful for the identification of the respective ligands oforphan receptors.

Since steroid receptors have three domains with different functions,which are more or less independent, it is possible that all threefunctional domains have been derived from different members of thesteroid receptor superfamily.

Molecules which contain parts having a different origin are calledchimeric. Such a chimeric receptor comprising the ligand-binding domainand/or the DNA-binding domain of the invention may be produced bychemical linkage, but most preferably the coupling is accomplished atthe DNA level with standard molecular biological methods by fusing thenucleic acid sequences encoding the necessary steroid receptor domains.Hence, DNA encoding the chimeric receptor proteins according to theinvention are also subject of the present invention.

Such chimeric proteins can be prepared by transfecting DNA encodingthese chimeric receptor proteins to suitable host cells and culturingthese cells under suitable conditions.

It is extremely practical if, next to the information for the expressionof the steroid receptor, also the host cell is transformed ortransfected with a vector which carries the information for a reportermolecule. Such a vector coding for a reporter molecule is characterizedby having a promoter sequence containing one or more hormone responsiveelements (HRE) functionally linked to an operative reporter gene. Such aHRE is the DNA target of the activated steroid receptor and, as aconsequence, it enhances the transcription of the DNA coding for thereporter molecule. In in vivo settings of steroid receptors the reportermolecule comprises the cellular response to the stimulation of theligand. However, it is possible in vitro to combine the ligand-bindingdomain of a receptor to the DNA binding domain and transcriptionactivating domain of other steroid receptors, thereby enabling the useof other HRE and reporter molecule systems. One such a system isestablished by a HRE presented in the MMTV-LTR (mouse mammary tumorvirus long terminal repeat sequence in connection with a reportermolecule like the firefly luciferase gene or the bacterial gene for CAT(chloramphenicol transferase). Other HRE's which can be used are the ratoxytocin promotor, the retinoic acid responsive element, the thyroidhormone responsive element, the estrogen responsive element and alsosynthetic responsive elements have been described (for instance inFuller, ibid. page 3096). As reporter molecules next to CAT andluciferase β-galactosidase can be used.

Steroid hormone receptors and chimeric receptors according to thepresent invention can be used for the in vitro identification of novelligands or hormonal analogs. For this purpose binding studies can beperformed with cells transformed with DNA according to the invention oran expression vector comprising DNA according to the invention, saidcells expressing the steroid receptors or chimeric receptors accordingto the invention.

The novel steroid hormone receptor and chimeric receptors according tothe invention as well as the ligand-binding domain of the invention, canbe used in an assay for the identification of functional ligands orhormone analogs for the nuclear receptors.

Thus, the present invention provides for a method for identifyingfunctional ligands for the steroid receptors and chimeric receptorsaccording to the invention, said method comprising the steps of

a) introducing into a suitable host cell 1) DNA or an expression vectoraccording to the invention, and 2) a suitable reporter gene functionallylinked to an operative hormone response element, said HRE being able tobe activated by the DNA-binding domain of the receptor protein encodedby said DNA;

b) bringing the host cell from step a) into contact with potentialligands which will possibly bind to the ligand-binding domain of thereceptor protein encoded by said DNA from step a);

c) monitoring the expression of the receptor protein encoded by saidreporter gene of step a).

If expression of the reporter gene is induced with respect to basicexpression (without ligand), the functional ligand can be considered asan agonist; if expression of the reporter gene remains unchanged or isreduced with respect to basic expression, the functional ligand can be asuitable (partial) antagonist.

For performing such kind of investigations host cells which have beentransformed or transfected with both a vector encoding a functionalsteroid receptor and a vector having the information for a hormoneresponsive element and a connected reporter molecule are cultured in asuitable medium. After addition of a suitable ligand, which willactivate the receptor the production of the reporter molecule will beenhanced, which production simply can be determined by assays having asensitivity for the reporter molecule. See for instance WO-A-8803168.Assays with known steroid receptors have been described (for instance S.Tsai et al., Cell 57, 443, 1989; M. Meyer et al., Cell 57, 433, 1989).

LEGENDS TO THE FIGURES

FIG. 1.

Northern analysis of the novel estrogen receptor (ERβ). Two differentmultiple tissue Northern blots (Clontech) were hybridised with aspecific probe for ERβ (see examples). Indicated are the human tissuesthe RNA originated from and the position of the size markers inkilobases (kb).

FIG. 2.

Histogram showing the 3- to 4-fold stimulatory effect of 17β-estradiol,estriol and estrone on the luciferase activity mediated by ERβ. Anexpression vector encoding ERβ was transiently transfected into CHOcells together with a reporter construct containing the rat oxytocinpromoter in front of the firefly luciferase encoding sequence (seeexamples).

FIG. 3.

Effect of 17β-estradiol (E2) alone or in combination with theanti-estrogen ICI-164384 (ICI) on ERβ and ERβ. Expression constructs forERα (the classical ER) and ERβ were transiently transfected into CHOcells together with the rat oxytocin promoter-luciferase reporterconstruct described in the examples. Luciferase activities weredetermined in triplicate and normalised for transfection efficiency bymeasuring β-galactosidase in the same lysate.

FIG. 4.

Expression of ERα and ERβ in a number of cell lines determined by RT-PCRanalysis (see examples). The cell lines used were derived from differenttissues/cell types: endometrium (ECC1, Ishikawa, HEC-1A, RL95-2);osteosarcoma (SAOS-2, U2-OS, HOS, MG63); breast tumours (MCF-7, T47D),endothelium (HUV-EC-C, BAEC-1); smooth muscle (HISM, PAC-1, A7R5, A10,RASMC, CavaSMC); liver (HepG2); colon (CaCo2); and vagina (Hs-760T,SW-954).

All cell lines were human except for PAC-1, A7R5, A10 and RASMC whichare of rat origin, BAEC-1 which is of bovine origin and CavaSMC which isof guinea pig origin.

FIG. 5.

Transactivation assay using stably transfected CHO cell lines expressingERα or ERβ together with the rat oxytocin-luciferase estrogen-responsivereporter (see examples for details). Hormone-dependent transactivationcurves were determined for 17β-estradiol and for Org4094. For the ERantagonist raloxifen, cells were treated with 2×10⁻¹⁰ mol/L17β-estradiol together with increasing concentrations of raloxifen.Maximal values of the responses were arbitrarily set at 100%.

EXAMPLES A. Molecular Cloning of the Novel Estrogen Receptor

Two degenerate oligonucleotides containing inosines (I) were based onconserved regions of the DNA-binding domains and the ligand-bindingdomains of the human steroid hormone receptors.

Primer #1:

5′-GGIGA(C/T)GA(A/G)GC(A/T)TCIGGITG(C/T)CA(C/T)TA(C/T)GG-3′ (SEQ IDNO:7).

Primer #2:

5′-AAGCCTGG(C/G)A(C/T)IC(G/T) (C/T)TTIGCCCAI(C/T)TIAT-3′ SEQ ID NO:8).

As template, CDNA from human EBV-stimulated PBLs (peripheral bloodleukocytes) was used. One microgram of total RNA was reverse transcribedin a 20 μl reaction containing 50 mM KCl, 10 mM Tris-HCl pH 8.3, 4 mMMgCl2, 1 mM dNTPs (Pharmacia), 100 pmol random hexanucleotides(Pharmacia), 30 Units RNAse inhibitor (Pharmacia) and 200 Units M-MLVReverse transcriptase (Gibco BRL). Reaction mixtures were incubated at37° C. for 30 minutes and heat-inactivated at 100° C. for 5 minutes. ThecDNA obtained was used in a 100 μl PCR reaction containing 10 mMTris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin (w/v), 3% DMSO,1 microgram of primer #1 and primer #2 and 2.5 Units of Amplitaq DNApolymerase (Perkin Elmer). PCR reactions were performed in the PerkinElmer 9600 thermal cycler. The initial denaturation (4 minutes at 94°C.) was followed by 35 cycles with the following conditions: 30 sec. 94°C., 30 sec. 45° C., 1 minute 72° C. and after 7 minutes at 72° C. thereactions were stored at 4° C. Aliquots of these reactions were analysedon a 1.5% agarose gel. Fragments of interest were cut out of the gel,reamplified using identical PCR-conditions and purified using Qiaex II(Qiagen). Fragments were cloned in the pCRII vector and transformed intobacteria using the TA-cloning kit (Invitrogen). Plasmid DNA was isolatedfor nucleotide sequence analysis using the Qiagen plasmid midi protocol(Qiagen). Nucleotide sequence analysis was performed with the ALFautomatic sequencer (Pharmacia) using a T7 DNA sequencing kit(Pharmacia) with vector-specific or fragment-specific primers.

One cloned fragment corresponded to a novel estrogen receptor (ER) whichis closely related to the classical estrogen receptor. Part of thecloned novel estrogen receptor fragment (nucleotides 466 to 797 in SEQID 1) was amplified by PCR using oligonucleotide #3TGTTACGAAGTGGGAATGGTGA (SEQ ID NO:9) and oligonucleotide #2 and used asa probe to screen a human testis cDNA library in λgt11 (Clontech#HL1010b). Recombinant phages were plated (using Y1090 bacteria grown inLB medium supplemented with 0.2% maltose) at a density of 40.000 pfu(plaque-forming units) per 135 mm dish and replica filters (Hybond-N,Amersham) were made as described by the supplier. Filters wereprehybridised in a solution containing 0.5 M phosphate buffer (pH 7.5)and 7% SDS at 65° C. for at least 30 minutes. DNA probes were purifiedwith Qiaex II (Qiagen), ³²P-labeled with a Decaprime kit (Ambion) andadded to the prehybridisation solution. Filters were hybridised at 65°C. overnight and then washed in 0.5×SSC/0.1% SDS at 65° C. Two positiveplaques were identified and could be shown to be identical. These cloneswere purified by rescreening one more time. A PCR reaction on the phageeluates with the λgt11-specific primers #4:5′-TTGACACCAGACCAACTGGTAATG-3′ (SEQ ID NO:10) and #5:5′-GGTGGCGACGACTCCTGGAGCCCG-3′ (SEQ ID NO:11) yielded a fragment of 1700basepairs on both clones. Subsequent PCR reactions using combinations ofa gene-specific primer #6: 5′-GTACACTGATTTGTAGCTGGAC-3′ (SEQ ID NO:12)with the λgtll primer #4 and gene-specific primer #7:5′-CCATGATGATGTCCCTGACC-3′ (SEQ ID NO:13) with λgt11 primer primer #5yielded fragments of approximately 450 bp and 1000 bp, respectively,which were cloned in the pCRII vector and used for nucleotide sequenceanalysis. The conditions for these PCR reactions were as described aboveexcept for the primer concentrations (200 ng of each primer) and theannealing temperature (60° C.). Since in the cDNA clone the homologywith the ER is lost abruptly at a site which corresponds to the exon7/exon 8 boundary in the ER (between nucleotides 1247 and 1248 in SEQ IDNO:1), it was suggested that this sequence corresponds to intron 7 ofthe novel ER gene. For verification of the nucleotide sequences of thiscDNA clone, a 1200 bp fragment was generated on the cDNA clone withλgt11 primer #4 with a gene-specific primer #8 corresponding to the 3′end of exon 7: 5′-TCGCATGCCTGACGTGGGAC-3′ (SEQ ID NO:14) using theproofreading Pfu polymerase (Stratagene). This fragment was also clonedin the pCRII vector and completely sequenced and was shown to beidentical to the sequences obtained earlier.

To obtain nucleotide sequences of the novel ER downstream of exon 7, adegenerate oligonucleotide based on the AF-2 region of the classical ER(#9: 5′-GGC(C/G)TCCAGCATCTCCAG(C/G)A(A/G)CAG-3′; SEQ ID NO:15) was usedtogether with the gene-specific oligonucleotide #10:5′-GGAAGCTGGCTCACTTGCTG-3′ (SEQ ID NO:16) using testis cDNA as template(Marathon ready testis cDNA, Clontech Cat #7414-1). A specific 220 bpfragment corresponding to nucleotides 1112 to 1332 in SEQ ID No. 1 wascloned and sequenced. Nucleotides 1112 to 1247 were identical to thecorresponding sequence of the cDNA clone. The sequence downstreamthereof is highly homologous with the corresponding region in theclassical ER. In order to obtain sequences of the novel ER downstream ofthe AF-2 region, RACE (rapid amplification of cDNA ends) PCR reactionswere performed using the Marathon-ready testis cDNA (Clontech) astemplate. The initial PCR was performed using oligonucleotide #11:5′-TCTTGTTCTGGACAGGGATG-3′ (SEQ ID NO:17) in combination with the AP1primer provided in the kit. A nested PCR was performed on an aliquot ofthis reaction using oligonucleotide #10 (SEQ ID NO:16) in combinationwith the oligo dT primer provided in the kit. Subsequently, an aliquotof this reaction was used in a nested PCR using oligonucleotide #12:5′-GCATGGAACATCTGCTCAAC-3′ (SEQ ID NO:18) in combination with the oligodT primer. Nucleotide sequence analysis of a specific fragment that wasobtained (corresponding to nucleotides 1256 to 1431 in SEQ ID NO 1)revealed a sequence encoding the carboxyterminus of the novel ERligand-binding domain, including an F-domain and a translational stopcodon and part of the 3′ untranslated sequence which is not included inSEQ ID NO:1. The deduced amino acid sequence is shown in SEQ ID NO:5.

In order to investigate the possibility that the novel estrogen receptorhad additional, upstream translation-initiation codons, RACE-PCRexperiments were performed using Marathon-ready testis cDNA (ClontechCat. #7414-1). First a PCR was performed using oligonucleotide SEQ IDNO:12 (antisense corresponding to nucleotides 416-395 in SEQ ID NO:1)and AP-1 (provided in the kit). A nested PCR was then performed usingoligonucleotide having SEQ ID NO:27 (antisense corresponding tonucleotides 254-231 in SEQ ID NO:1) with AP-2 (provided in the kit).From the smear that was obtained, the region corresponding to fragmentslarger than 300 basepairs was cut out, purified using the GenecleanIIkit (Bio101) and cloned using the TA-cloning kit (Clontech). Colonieswere screened by PCR using gene-specific primers: SEQ ID NO:22 and SEQID NO:28. The clone containing the largest insert was sequenced. Thenucleotide sequence corresponds to nucleotides 1 to 490 in SEQ ID NO:24.It is clear from this sequence that the first in-frame upstreamtranslation initiation codon is present at position 77-79 in SEQ IDNO:24. Upstream of this translational startcodon an in-frame stop-codonis present (11-13 in SEQ ID NO:24). Consequently, the reading frame ofthe novel estrogen receptor is 530 amino acids (shown in SEQ ID NO:25)and has a calculated molecular mass of 59.234 kD.

To confirm the nucleotide sequences obtained by 5′ RACE, human genomicclones were obtained and analysed. A human genomic library in λEMBL3(Clontech HL1067J) was screened with a probe corresponding tonucleotides 1 to 416 in SEQ ID NO:1. A strongly hybridizing clone wasplaque-purified and DNA was isolated using standard protocols (Sambrooket al, 1989). The DNA was digested with several restriction enzymes,electrophoresed on agarose gel and blotted onto Nylon filters.Hybridisation of the blot with a probe corresponding to theabove-mentioned RACE fragment (nucleotides 1-490 in SEQ ID NO:24)revealed a hybridizing Sau3A fragment of approximately 800 basepairs.This fragment was cloned into the BamH1 site of pGEM3Z and sequenced.The nucleotide sequence contained one base difference which is probablya PCR-induced point mutation in the RACE fragment. Nucleotide 172 was aG residue in the 5′RACE fragment, but an A residue in severalindependent genomic subclones.

B. Identification of Two Splice Variants of the Novel Estrogen Receptor

Rescreening of the testis cDNA library with a probe corresponding tonucleotides 918 to 1246 in SEQ ID No. 1 yielded two hybridizing clones,the 3′ end of which were amplified by PCR (gene-specific primer #10:5′-GGAAGCTGGCTCACTTGCTG-3′ (SEQ ID NO:16) together with primer #4, SEQID NO:10), cloned and sequenced. One clone was shown to contain analternative exon 8 (exon 8B) of the novel ER. In SEQ ID No. 2 theprotein encoding part and the stopcodon of this splice variant arepresented. As a consequence of the introduction of this exon through analternative splicing reaction, the reading frame encoding the novel ERis immediately terminated, thereby creating a truncation of thecarboxyterminus of the novel ER (SEQ ID NO:6).

Screening of a human thymus CDNA library (Clontech HL1074a) with theprobe corresponding to nucleotides 918 to 1246 in SEQ ID No. 1, revealedanother splice variant. The 3′ end of one hybridizing clone wasamplified using primer #10 (SEQ ID NO:16) with the λgt10-specific primer#13 5′-AGCAAGTTCAGCCTGTTAAGT-3′ (SEQ ID NO:19), cloned and sequenced.The obtained nucleotide sequence upstream of the exon 7/exon 8 boundarywas identical to the clones identified earlier. However, an alternativeexon 8 (exon 8C) was present at the 3′ end encoding two C-terminal aminoacids followed by a stop-codon. The nucleotide sequence of theprotein-encoding part of this splice variant is shown in SEQ ID NO:20,the corresponding protein sequence is SEQ ID NO:21.

These two variants of the novel estrogen receptor do not contain theAF-2 region and therefore probably lack the ability to modulatetranscription of target genes in a ligand-dependent fashion. However,the variants potentially could interfere with the functioning of thewild-type classical ER and/or the wild-type novel ER, either byheterodimerization or by occupying estrogen response elements or byinteractions with other transcription factors. A mutant of the classicalER (ER1-530) has been described which closely resembles the two variantsof the novel estrogen receptor described above. ER1-530 has been shownto behave as a dominant-negative receptor 25 i.e. it can modulate theintracellular activity of the wild type ER (Ince et al, J. Biol. Chem.268, 14026-14032, 1993).

C. Northern Blot Analysis

Human multiple tissue Northern blots (MTN-blots) were purchased fromClontech and prehybridized for at least 1 hour at 65° C. in 0.5 Mphosphate buffer pH 7.5 with 7% SDS. The DNA fragment that was used as aprobe (corresponding to nucleotides 466 to 797 in SEQ ID No. 1) was³²P-labeled using a labelling kit (Ambion), denatured by boiling andadded to the prehybridisation solution. Washing conditions were: 3×SSCat room temperature, followed by 3×SSC at 65° C., and finally 1×SSC at65° C. The filters were than exposed to X-ray films for one week. Twotranscripts of approximately 8 kb and 10 kb were detected in thymus,spleen, ovary and testis. In addition, a 1.3 kb transcript was detectedin testis.

D. RT-PCR Analysis of Expression of ERα and ERβ in Cell Lines

RNA was isolated from a number of human and animal cell lines usingRNAzol B (Cinna/Biotecx). cDNA was made using 2.5 microgram of total RNAusing the Superscript II kit (BRL) following the manufacturersinstructions. A portion of the cDNA was used for specific PCRamplifications of fragments corresponding either to mRNA encoding the ERor to the novel estrogen receptor. (It should be emphasized that theprimers used are based on human and rat sequences, whereas some of thecell lines were not rat or human, see legend of FIG. 4). Primers usedwere for ERα: sense 5′-GATGGGCTTACTGACCAACC-3′ and antisense5′-AGATGCTCCATGCCTTTG-3′ generating a 548 base pair fragmentcorresponding to part of the LBD. For ERβ: sense5′-TTCACCGAGGCCTCCATGATG-3′ and antisense 5′-CAGATGTTCCATGCCCTTGTT-3′generating a 565 base pair fragment corresponding to part of the LBD.The PCR samples were analysed on agarose which were blotted onto Nylonmembranes. These blots were hybridised with ³²P-labeled PCR fragmentsgenerated with the above-mentioned primers on ERα and ERβ plasmid DNAusing standard experimental procedures (Sambrook et al, 1989).

E. Ligand-dependent Transcription Activation by the Novel EstrogenReceptor Protein

Cell Culture

Chinese Hamster Ovary (CHO K1) cells were obtained from ATCC (CCL61) andmaintained at 37° C. in a humidified atmosphere (5% CO₂) as a monolayerculture in fenolred-free M505 medium. The latter medium consists of amixture (1:1) of Dulbecco's Modified Eagle's Medium (DMEM, Gibco074-200) and Nutrient Medium F12 (Ham's F12, Gibco 074-1700)supplemented with 2.5 mg/ml sodium carbonate (Baker), 55 μg/ml sodiumpyruvate (Fluka), 2.3 μg/ml β-mercaptoethanol (Baker), 1.2 μg/mlethanolamine (Baker), 360 μg/ml L-glutamine (Merck), 0.45 μg/ml sodiumselenite (Fluka), 62.5 μg/ml penicillin (Mycopharm), 62.5 μg/mlstreptomycin (Serva), and 5% charcoal-treated bovine calf serum(Hyclone).

Recombinant Vectors

The ERβ-encoding sequence as presented in SEQ ID No. 1 was amplified byPCR using oligonucleotides 5′-CTTGGATCCATAGCCCTGCTGTGATGAATTACAG-3′ (SEQID NO:22 underlined is the translation initiation codon) in combinationwith 5′-GATGGATCCTCACCTCAGGGCCAGGCGTCACTG-3′ (SEQ ID NO:23) (underlinedis the translation stopcodon, antisense). The resulting BamH1 fragment(approximately 1450 base pairs) were then cloned in the mammalian cellexpression vector pNGV1 (Genbank accession No. X99274).

An expression construct encoding the ERβ reading frame as presented inSEQ ID NO:24 was made by replacing a BamH1-Msc1 fragment (nucleotides1-81 in SEQ ID No. 1) by a BamH1-Msc1 fragment corresponding tonucleotides 77-316 in SEQ ID No. 24. The latter fragment was made by PCRwith SEQ ID NO:26 in combination with SEQ ID NO:28 using the abovementioned 5′ RACE fragment.

The reporter vector was based on the rat oxytocin gene regulatory region(position −363/+16 as a HindIII/MboI fragment; R. Ivell, and D. Richter,Proc.Natl.Acad.Sci.USA 81, 2006-2010, 1984) linked to the fireflyluciferase encoding sequence; the regulatory region of the oxytocin genewas shown to possess functional estrogen hormone response elements invitro for both the rat (R. Adan et al, Biochem.Biophys.Res.Comm. 175,117-122, 1991) and the human (S. Richard, and H. Zingg, J.Biol.Chem.265, 6098-6103, 1990).

Transient Transfection

1×10⁵CHO cells were seeded in 6-wells Nunclon tissue culture plates andDNA was introduced by use of lipofectin (Gibco BRL). Hereto, the DNA (1μg of both receptor and reporter vector in 250 μL Optimem, Gibco BRL)was mixed with an equal volume of lipofectin reagent (7 μL in 250 μLOptimem, Gibco) and allowed to stand at room temperature for 15 min.After washing the cells twice with serum-free medium (M505) new medium(500 μL Optimem, Gibco) was added to the cells followed by the dropwiseaddition of the DNA-lipofectin mixture. After incubation for a 5 hourperiod at 37° C. cells were washed twice with fenolred-free M505+5%charcoal-treated bovine calf serum and incubated overnight at 37° C.After 24 hours hormones were added to the medium (10⁻⁷ mol/L). Cellextracts were made 48 hours posttransfection by the addition of 200 μLlysisbuffer (0.1 M phosphate buffer pH7.8, 0.2% Triton X-100). Afterincubation for 5 min at 37° C. the cell suspension was centrifuged(Eppendorf centrifuge, 5 min) and 20 μL sample was added to 50 μLluciferase assay reagent (Promega). Light emission was measured in aluminometer (Berthold Biolumat) for 10 sec at 562 nm.

Stable Transfection of the Novel Estrogen Receptor.

The expression plasmid encoding full-length ERβ1-530 (see above) wasstably transfected in CHO K1 cells as previously described (Theunissenet al., J. Biol. Chem. 268, 9035-9040, 1993). Single cell clones thatwere obtained this way were screened by transient transfection of thereporter plasmid (rat oxytocin-luciferase) as described above. Selectedclones were used for a second stable transfection of the ratoxytocin-luciferase reporter plasmid together with the plasmid pDR2Awhich contains a hygromycine resitance gene for selection. Single cellclones obtained were tested for a response to 17β-estradiol.Subsequently, a selected single cell clone was used for transactivationstudies. Briefly, cells were seeded in 96-wells at (1.6×10⁴ cells perwell) After 24 hours different concentrations of hormone were diluted inmedium and added to the wells. For antagonistic experiments, 2×10⁻¹⁰ M.17β-estradiol was added to each well and different concentrations ofantagonists were added. Cells were washed once with PBS after a 24 hourincubation and then lysed by the addition of 40 microliter lysis buffer(see above). Luciferase reagent was added (50 microliter) to each welland light emission was measured using the Topcount (Packard).

Results.

A comparison of the two expression constructs (SEQ ID NO:1 and SEQ IDNO:24) in transient transfections in CHO cells showed identicaltransactivation in response to a number of agonists and antagonists. CHOcells transiently transfected with ERβ expression vector and a reporterplasmid showed a 3 to 4 fold increase in luciferase activity in responseto 17β-estradiol as compared to untreated cells (see FIG. 2). A similartransactivation was obtained upon treatment with estriol and estrone.The results indicate not only that the novel ER (ERβ) can bind estrogenhormones but also that the ligand-activated receptor can bind to theestrogen-response elements (EREs) within the rat oxytocin promoter andactivate transcription of the luciferase reporter gene. FIG. 3 showsthat in an independent similar experiment 10⁻⁹ mol/L 17β-estradiol gavean 18-fold stimulation with ERα and a 7-fold stimulation with ERβ. Inaddition, the antiestrogen ICI-164384 was shown to be an antagonist forboth ERα and ERβ when activated with 17β-estradiol, whereas theantagonist alone had no effect. In this experiment 0.25 μgβ-galactosidase vector was co-transfected in order to normalize fordifferences in transfection efficiency.

Transactivation studies performed on stably transfected ERα and ERβ celllines gave similar absolute luciferase values. The curves for17β-estradiol are very similar and show that half-maximaltransactivation is reached with lower concentrations of hormone on ERαas compared to ERβ (FIG. 5). For Org4094 this is also the case however,the effect observed is much more pronounced. The curves for raloxifenshow that the potency of this antagonist to block transactivation on ERαis greater compared to its potency to block ERβ transactivation.

28 1 1434 DNA Homo sapiens 1 atgaattaca gcattcccag caatgtcact aacttggaaggtgggcctgg tcggcagacc 60 acaagcccaa atgtgttgtg gccaacacct gggcacctttctcctttagt ggtccatcgc 120 cagttatcac atctgtatgc ggaacctcaa aagagtccctggtgtgaagc aagatcgcta 180 gaacacacct tacctgtaaa cagagagaca ctgaaaaggaaggttagtgg gaaccgttgc 240 gccagccctg ttactggtcc aggttcaaag agggatgctcacttctgcgc tgtctgcagc 300 gattacgcat cgggatatca ctatggagtc tggtcgtgtgaaggatgtaa ggcctttttt 360 aaaagaagca ttcaaggaca taatgattat atttgtccagctacaaatca gtgtacaatc 420 gataaaaacc ggcgcaagag ctgccaggcc tgccgacttcggaagtgtta cgaagtggga 480 atggtgaagt gtggctcccg gagagagaga tgtgggtaccgccttgtgcg gagacagaga 540 agtgccgacg agcagctgca ctgtgccggc aaggccaagagaagtggcgg ccacgcgccc 600 cgagtgcggg agctgctgct ggacgccctg agccccgagcagctagtgct caccctcctg 660 gaggctgagc cgccccatgt gctgatcagc cgccccagtgcgcccttcac cgaggcctcc 720 atgatgatgt ccctgaccaa gttggccgac aaggagttggtacacatgat cagctgggcc 780 aagaagattc ccggctttgt ggagctcagc ctgttcgaccaagtgcggct cttggagagc 840 tgttggatgg aggtgttaat gatggggctg atgtggcgctcaattgacca ccccggcaag 900 ctcatctttg ctccagatct tgttctggac agggatgaggggaaatgcgt agaaggaatt 960 ctggaaatct ttgacatgct cctggcaact acttcaaggtttcgagagtt aaaactccaa 1020 cacaaagaat atctctgtgt caaggccatg atcctgctcaattccagtat gtaccctctg 1080 gtcacagcga cccaggatgc tgacagcagc cggaagctggctcacttgct gaacgccgtg 1140 accgatgctt tggtttgggt gattgccaag agcggcatctcctcccagca gcaatccatg 1200 cgcctggcta acctcctgat gctcctgtcc cacgtcaggcatgcgagtaa caagggcatg 1260 gaacatctgc tcaacatgaa gtgcaaaaat gtggtcccagtgtatgacct gctgctggag 1320 atgctgaatg cccacgtgct tcgcgggtgc aagtcctccatcacggggtc cgagtgcagc 1380 ccggcagagg acagtaaaag caaagagggc tcccagaacccacagtctca gtga 1434 2 1251 DNA Homo sapiens 2 atgaattaca gcattcccagcaatgtcact aacttggaag gtgggcctgg tcggcagacc 60 acaagcccaa atgtgttgtggccaacacct gggcaccttt ctcctttagt ggtccatcgc 120 cagttatcac atctgtatgcggaacctcaa aagagtccct ggtgtgaagc aagatcgcta 180 gaacacacct tacctgtaaacagagagaca ctgaaaagga aggttagtgg gaaccgttgc 240 gccagccctg ttactggtccaggttcaaag agggatgctc acttctgcgc tgtctgcagc 300 gattacgcat cgggatatcactatggagtc tggtcgtgtg aaggatgtaa ggcctttttt 360 aaaagaagca ttcaaggacataatgattat atttgtccag ctacaaatca gtgtacaatc 420 gataaaaacc ggcgcaagagctgccaggcc tgccgacttc ggaagtgtta cgaagtggga 480 atggtgaagt gtggctcccggagagagaga tgtgggtacc gccttgtgcg gagacagaga 540 agtgccgacg agcagctgcactgtgccggc aaggccaaga gaagtggcgg ccacgcgccc 600 cgagtgcggg agctgctgctggacgccctg agccccgagc agctagtgct caccctcctg 660 gaggctgagc cgccccatgtgctgatcagc cgccccagtg cgcccttcac cgaggcctcc 720 atgatgatgt ccctgaccaagttggccgac aaggagttgg tacacatgat cagctgggcc 780 aagaagattc ccggctttgtggagctcagc ctgttcgacc aagtgcggct cttggagagc 840 tgttggatgg aggtgttaatgatggggctg atgtggcgct caattgacca ccccggcaag 900 ctcatctttg ctccagatcttgttctggac agggatgagg ggaaatgcgt agaaggaatt 960 ctggaaatct ttgacatgctcctggcaact acttcaaggt ttcgagagtt aaaactccaa 1020 cacaaagaat atctctgtgtcaaggccatg atcctgctca attccagtat gtaccctctg 1080 gtcacagcga cccaggatgctgacagcagc cggaagctgg ctcacttgct gaacgccgtg 1140 accgatgctt tggtttgggtgattgccaag agcggcatct cctcccagca gcaatccatg 1200 cgcctggcta acctcctgatgctcctgtcc cacgtcaggc atgcgaggtg a 1251 3 66 PRT Homo sapiens 3 Cys AlaVal Cys Ser Asp Tyr Ala Ser Gly Tyr His Tyr Gly Val Trp 1 5 10 15 SerCys Glu Gly Cys Lys Ala Phe Phe Lys Arg Ser Ile Gln Gly His 20 25 30 AsnAsp Tyr Ile Cys Pro Ala Thr Asn Gln Cys Thr Ile Asp Lys Asn 35 40 45 ArgArg Lys Ser Cys Gln Ala Cys Arg Leu Arg Lys Cys Tyr Glu Val 50 55 60 GlyMet 65 4 233 PRT Homo sapiens 4 Leu Val Leu Thr Leu Leu Glu Ala Glu ProPro His Val Leu Ile Ser 1 5 10 15 Arg Pro Ser Ala Pro Phe Thr Glu AlaSer Met Met Met Ser Leu Thr 20 25 30 Lys Leu Ala Asp Lys Glu Leu Val HisMet Ile Ser Trp Ala Lys Lys 35 40 45 Ile Pro Gly Phe Val Glu Leu Ser LeuPhe Asp Gln Val Arg Leu Leu 50 55 60 Glu Ser Cys Trp Met Glu Val Leu MetMet Gly Leu Met Trp Arg Ser 65 70 75 80 Ile Asp His Pro Gly Lys Leu IlePhe Ala Pro Asp Leu Val Leu Asp 85 90 95 Arg Asp Glu Gly Lys Cys Val GluGly Ile Leu Glu Ile Phe Asp Met 100 105 110 Leu Leu Ala Thr Thr Ser ArgPhe Arg Glu Leu Lys Leu Gln His Lys 115 120 125 Glu Tyr Leu Cys Val LysAla Met Ile Leu Leu Asn Ser Ser Met Tyr 130 135 140 Pro Leu Val Thr AlaThr Gln Asp Ala Asp Ser Ser Arg Lys Leu Ala 145 150 155 160 His Leu LeuAsn Ala Val Thr Asp Ala Leu Val Trp Val Ile Ala Lys 165 170 175 Ser GlyIle Ser Ser Gln Gln Gln Ser Met Arg Leu Ala Asn Leu Leu 180 185 190 MetLeu Leu Ser His Val Arg His Ala Ser Asn Lys Gly Met Glu His 195 200 205Leu Leu Asn Met Lys Cys Lys Asn Val Val Pro Val Tyr Asp Leu Leu 210 215220 Leu Glu Met Leu Asn Ala His Val Leu 225 230 5 477 PRT Homo sapiens 5Met Asn Tyr Ser Ile Pro Ser Asn Val Thr Asn Leu Glu Gly Gly Pro 1 5 1015 Gly Arg Gln Thr Thr Ser Pro Asn Val Leu Trp Pro Thr Pro Gly His 20 2530 Leu Ser Pro Leu Val Val His Arg Gln Leu Ser His Leu Tyr Ala Glu 35 4045 Pro Gln Lys Ser Pro Trp Cys Glu Ala Arg Ser Leu Glu His Thr Leu 50 5560 Pro Val Asn Arg Glu Thr Leu Lys Arg Lys Val Ser Gly Asn Arg Cys 65 7075 80 Ala Ser Pro Val Thr Gly Pro Gly Ser Lys Arg Asp Ala His Phe Cys 8590 95 Ala Val Cys Ser Asp Tyr Ala Ser Gly Tyr His Tyr Gly Val Trp Ser100 105 110 Cys Glu Gly Cys Lys Ala Phe Phe Lys Arg Ser Ile Gln Gly HisAsn 115 120 125 Asp Tyr Ile Cys Pro Ala Thr Asn Gln Cys Thr Ile Asp LysAsn Arg 130 135 140 Arg Lys Ser Cys Gln Ala Cys Arg Leu Arg Lys Cys TyrGlu Val Gly 145 150 155 160 Met Val Lys Cys Gly Ser Arg Arg Glu Arg CysGly Tyr Arg Leu Val 165 170 175 Arg Arg Gln Arg Ser Ala Asp Glu Gln LeuHis Cys Ala Gly Lys Ala 180 185 190 Lys Arg Ser Gly Gly His Ala Pro ArgVal Arg Glu Leu Leu Leu Asp 195 200 205 Ala Leu Ser Pro Glu Gln Leu ValLeu Thr Leu Leu Glu Ala Glu Pro 210 215 220 Pro His Val Leu Ile Ser ArgPro Ser Ala Pro Phe Thr Glu Ala Ser 225 230 235 240 Met Met Met Ser LeuThr Lys Leu Ala Asp Lys Glu Leu Val His Met 245 250 255 Ile Ser Trp AlaLys Lys Ile Pro Gly Phe Val Glu Leu Ser Leu Phe 260 265 270 Asp Gln ValArg Leu Leu Glu Ser Cys Trp Met Glu Val Leu Met Met 275 280 285 Gly LeuMet Trp Arg Ser Ile Asp His Pro Gly Lys Leu Ile Phe Ala 290 295 300 ProAsp Leu Val Leu Asp Arg Asp Glu Gly Lys Cys Val Glu Gly Ile 305 310 315320 Leu Glu Ile Phe Asp Met Leu Leu Ala Thr Thr Ser Arg Phe Arg Glu 325330 335 Leu Lys Leu Gln His Lys Glu Tyr Leu Cys Val Lys Ala Met Ile Leu340 345 350 Leu Asn Ser Ser Met Tyr Pro Leu Val Thr Ala Thr Gln Asp AlaAsp 355 360 365 Ser Ser Arg Lys Leu Ala His Leu Leu Asn Ala Val Thr AspAla Leu 370 375 380 Val Trp Val Ile Ala Lys Ser Gly Ile Ser Ser Gln GlnGln Ser Met 385 390 395 400 Arg Leu Ala Asn Leu Leu Met Leu Leu Ser HisVal Arg His Ala Ser 405 410 415 Asn Lys Gly Met Glu His Leu Leu Asn MetLys Cys Lys Asn Val Val 420 425 430 Pro Val Tyr Asp Leu Leu Leu Glu MetLeu Asn Ala His Val Leu Arg 435 440 445 Gly Cys Lys Ser Ser Ile Thr GlySer Glu Cys Ser Pro Ala Glu Asp 450 455 460 Ser Lys Ser Lys Glu Gly SerGln Asn Pro Gln Ser Gln 465 470 475 6 416 PRT Homo sapiens 6 Met Asn TyrSer Ile Pro Ser Asn Val Thr Asn Leu Glu Gly Gly Pro 1 5 10 15 Gly ArgGln Thr Thr Ser Pro Asn Val Leu Trp Pro Thr Pro Gly His 20 25 30 Leu SerPro Leu Val Val His Arg Gln Leu Ser His Leu Tyr Ala Glu 35 40 45 Pro GlnLys Ser Pro Trp Cys Glu Ala Arg Ser Leu Glu His Thr Leu 50 55 60 Pro ValAsn Arg Glu Thr Leu Lys Arg Lys Val Ser Gly Asn Arg Cys 65 70 75 80 AlaSer Pro Val Thr Gly Pro Gly Ser Lys Arg Asp Ala His Phe Cys 85 90 95 AlaVal Cys Ser Asp Tyr Ala Ser Gly Tyr His Tyr Gly Val Trp Ser 100 105 110Cys Glu Gly Cys Lys Ala Phe Phe Lys Arg Ser Ile Gln Gly His Asn 115 120125 Asp Tyr Ile Cys Pro Ala Thr Asn Gln Cys Thr Ile Asp Lys Asn Arg 130135 140 Arg Lys Ser Cys Gln Ala Cys Arg Leu Arg Lys Cys Tyr Glu Val Gly145 150 155 160 Met Val Lys Cys Gly Ser Arg Arg Glu Arg Cys Gly Tyr ArgLeu Val 165 170 175 Arg Arg Gln Arg Ser Ala Asp Glu Gln Leu His Cys AlaGly Lys Ala 180 185 190 Lys Arg Ser Gly Gly His Ala Pro Arg Val Arg GluLeu Leu Leu Asp 195 200 205 Ala Leu Ser Pro Glu Gln Leu Val Leu Thr LeuLeu Glu Ala Glu Pro 210 215 220 Pro His Val Leu Ile Ser Arg Pro Ser AlaPro Phe Thr Glu Ala Ser 225 230 235 240 Met Met Met Ser Leu Thr Lys LeuAla Asp Lys Glu Leu Val His Met 245 250 255 Ile Ser Trp Ala Lys Lys IlePro Gly Phe Val Glu Leu Ser Leu Phe 260 265 270 Asp Gln Val Arg Leu LeuGlu Ser Cys Trp Met Glu Val Leu Met Met 275 280 285 Gly Leu Met Trp ArgSer Ile Asp His Pro Gly Lys Leu Ile Phe Ala 290 295 300 Pro Asp Leu ValLeu Asp Arg Asp Glu Gly Lys Cys Val Glu Gly Ile 305 310 315 320 Leu GluIle Phe Asp Met Leu Leu Ala Thr Thr Ser Arg Phe Arg Glu 325 330 335 LeuLys Leu Gln His Lys Glu Tyr Leu Cys Val Lys Ala Met Ile Leu 340 345 350Leu Asn Ser Ser Met Tyr Pro Leu Val Thr Ala Thr Gln Asp Ala Asp 355 360365 Ser Ser Arg Lys Leu Ala His Leu Leu Asn Ala Val Thr Asp Ala Leu 370375 380 Val Trp Val Ile Ala Lys Ser Gly Ile Ser Ser Gln Gln Gln Ser Met385 390 395 400 Arg Leu Ala Asn Leu Leu Met Leu Leu Ser His Val Arg HisAla Arg 405 410 415 7 29 DNA Homo sapiens misc_feature (1)..(29) n isinosine 7 ggngaygarg cwtcnggntg ycaytaygg 29 8 29 DNA Homo sapiensmisc_feature (1)..(29) n is inosine 8 aacggtggsa ynckyttngc ccanytnat 299 22 DNA Homo sapiens 9 tgttacgaag tgggaatggt ga 22 10 24 DNA Homosapiens 10 ttgacaccag accaactggt aatg 24 11 24 DNA Homo sapiens 11ggtggcgacg actcctggag cccg 24 12 22 DNA Homo sapiens 12 gtacactgatttgtagctgg ac 22 13 20 DNA Homo sapiens 13 ccatgatgat gtccctgacc 20 1420 DNA Homo sapiens 14 tcgcatgcct gacgtgggac 20 15 24 DNA Homo sapiens15 ggcstccagc atctccagsa rcag 24 16 20 DNA Homo sapiens 16 ggaagctggctcacttgctg 20 17 20 DNA Homo sapiens 17 tcttgttctg gacagggatg 20 18 20DNA Homo sapiens 18 gcatggaaca tctgctcaac 20 19 21 DNA Homo sapiens 19agcaagttca gcctgttaag t 21 20 1257 DNA Homo sapiens 20 atgaattacagcattcccag caatgtcact aacttggaag gtgggcctgg tcggcagacc 60 acaagcccaaatgtgttgtg gccaacacct gggcaccttt ctcctttagt ggtccatcgc 120 cagttatcacatctgtatgc ggaacctcaa aagagtccct ggtgtgaagc aagatcgcta 180 gaacacaccttacctgtaaa cagagagaca ctgaaaagga aggttagtgg gaaccgttgc 240 gccagccctgttactggtcc aggttcaaag agggatgctc acttctgcgc tgtctgcagc 300 gattacgcatcgggatatca ctatggagtc tggtcgtgtg aaggatgtaa ggcctttttt 360 aaaagaagcattcaaggaca taatgattat atttgtccag ctacaaatca gtgtacaatc 420 gataaaaaccggcgcaagag ctgccaggcc tgccgacttc ggaagtgtta cgaagtggga 480 atggtgaagtgtggctcccg gagagagaga tgtgggtacc gccttgtgcg gagacagaga 540 agtgccgacgagcagctgca ctgtgccggc aaggccaaga gaagtggcgg ccacgcgccc 600 cgagtgcgggagctgctgct ggacgccctg agccccgagc agctagtgct caccctcctg 660 gaggctgagccgccccatgt gctgatcagc cgccccagtg cgcccttcac cgaggcctcc 720 atgatgatgtccctgaccaa gttggccgac aaggagttgg tacacatgat cagctgggcc 780 aagaagattcccggctttgt ggagctcagc ctgttcgacc aagtgcggct cttggagagc 840 tgttggatggaggtgttaat gatggggctg atgtggcgct caattgacca ccccggcaag 900 ctcatctttgctccagatct tgttctggac agggatgagg ggaaatgcgt agaaggaatt 960 ctggaaatctttgacatgct cctggcaact acttcaaggt ttcgagagtt aaaactccaa 1020 cacaaagaatatctctgtgt caaggccatg atcctgctca attccagtat gtaccctctg 1080 gtcacagcgacccaggatgc tgacagcagc cggaagctgg ctcacttgct gaacgccgtg 1140 accgatgctttggtttgggt gattgccaag agcggcatct cctcccagca gcaatccatg 1200 cgcctggctaacctcctgat gctcctgtcc cacgtcaggc atgcgaggtc tgcctga 1257 21 418 PRT Homosapiens 21 Met Asn Tyr Ser Ile Pro Ser Asn Val Thr Asn Leu Glu Gly GlyPro 1 5 10 15 Gly Arg Gln Thr Thr Ser Pro Asn Val Leu Trp Pro Thr ProGly His 20 25 30 Leu Ser Pro Leu Val Val His Arg Gln Leu Ser His Leu TyrAla Glu 35 40 45 Pro Gln Lys Ser Pro Trp Cys Glu Ala Arg Ser Leu Glu HisThr Leu 50 55 60 Pro Val Asn Arg Glu Thr Leu Lys Arg Lys Val Ser Gly AsnArg Cys 65 70 75 80 Ala Ser Pro Val Thr Gly Pro Gly Ser Lys Arg Asp AlaHis Phe Cys 85 90 95 Ala Val Cys Ser Asp Tyr Ala Ser Gly Tyr His Tyr GlyVal Trp Ser 100 105 110 Cys Glu Gly Cys Lys Ala Phe Phe Lys Arg Ser IleGln Gly His Asn 115 120 125 Asp Tyr Ile Cys Pro Ala Thr Asn Gln Cys ThrIle Asp Lys Asn Arg 130 135 140 Arg Lys Ser Cys Gln Ala Cys Arg Leu ArgLys Cys Tyr Glu Val Gly 145 150 155 160 Met Val Lys Cys Gly Ser Arg ArgGlu Arg Cys Gly Tyr Arg Leu Val 165 170 175 Arg Arg Gln Arg Ser Ala AspGlu Gln Leu His Cys Ala Gly Lys Ala 180 185 190 Lys Arg Ser Gly Gly HisAla Pro Arg Val Arg Glu Leu Leu Leu Asp 195 200 205 Ala Leu Ser Pro GluGln Leu Val Leu Thr Leu Leu Glu Ala Glu Pro 210 215 220 Pro His Val LeuIle Ser Arg Pro Ser Ala Pro Phe Thr Glu Ala Ser 225 230 235 240 Met MetMet Ser Leu Thr Lys Leu Ala Asp Lys Glu Leu Val His Met 245 250 255 IleSer Trp Ala Lys Lys Ile Pro Gly Phe Val Glu Leu Ser Leu Phe 260 265 270Asp Gln Val Arg Leu Leu Glu Ser Cys Trp Met Glu Val Leu Met Met 275 280285 Gly Leu Met Trp Arg Ser Ile Asp His Pro Gly Lys Leu Ile Phe Ala 290295 300 Pro Asp Leu Val Leu Asp Arg Asp Glu Gly Lys Cys Val Glu Gly Ile305 310 315 320 Leu Glu Ile Phe Asp Met Leu Leu Ala Thr Thr Ser Arg PheArg Glu 325 330 335 Leu Lys Leu Gln His Lys Glu Tyr Leu Cys Val Lys AlaMet Ile Leu 340 345 350 Leu Asn Ser Ser Met Tyr Pro Leu Val Thr Ala ThrGln Asp Ala Asp 355 360 365 Ser Ser Arg Lys Leu Ala His Leu Leu Asn AlaVal Thr Asp Ala Leu 370 375 380 Val Trp Val Ile Ala Lys Ser Gly Ile SerSer Gln Gln Gln Ser Met 385 390 395 400 Arg Leu Ala Asn Leu Leu Met LeuLeu Ser His Val Arg His Ala Arg 405 410 415 Ser Ala 22 34 DNA Homosapiens 22 cttggatcca tagccctgct gtgatgaatt acag 34 23 33 DNA Homosapiens 23 gatggatcct cacctcaggg ccaggcgtca ctg 33 24 1898 DNA Homosapiens 24 cacgaatctt tgagaacatt ataatgacct ttgtgcctct tcttgcaaggtgttttctca 60 gctgttatct caagacatgg atataaaaaa ctcaccatct agccttaattctccttcctc 120 ctacaactgc agtcaatcca tcttacccct ggagcacggc tccatatacataccttcctc 180 ctatgtagac agccaccatg aatatccagc catgacattc tatagccctgctgtgatgaa 240 ttacagcatt cccagcaatg tcactaactt ggaaggtggg cctggtcggcagaccacaag 300 cccaaatgtg ttgtggccaa cacctgggca cctttctcct ttagtggtccatcgccagtt 360 atcacatctg tatgcggaac ctcaaaagag tccctggtgt gaagcaagatcgctagaaca 420 caccttacct gtaaacagag agacactgaa aaggaaggtt agtgggaaccgttgcgccag 480 ccctgttact ggtccaggtt caaagaggga tgctcacttc tgcgctgtctgcagcgatta 540 cgcatcggga tatcactatg gagtctggtc gtgtgaagga tgtaaggccttttttaaaag 600 aagcattcaa ggacataatg attatatttg tccagctaca aatcagtgtacaatcgataa 660 aaaccggcgc aagagctgcc aggcctgccg acttcggaag tgttacgaagtgggaatggt 720 gaagtgtggc tcccggagag agagatgtgg gtaccgcctt gtgcggagacagagaagtgc 780 cgacgagcag ctgcactgtg ccggcaaggc caagagaagt ggcggccacgcgccccgagt 840 gcgggagctg ctgctggacg ccctgagccc cgagcagcta gtgctcaccctcctggaggc 900 tgagccgccc catgtgctga tcagccgccc cagtgcgccc ttcaccgaggcctccatgat 960 gatgtccctg accaagttgg ccgacaagga gttggtacac atgatcagctgggccaagaa 1020 gattcccggc tttgtggagc tcagcctgtt cgaccaagtg cggctcttggagagctgttg 1080 gatggaggtg ttaatgatgg ggctgatgtg gcgctcaatt gaccaccccggcaagctcat 1140 ctttgctcca gatcttgttc tggacaggga tgaggggaaa tgcgtagaaggaattctgga 1200 aatctttgac atgctcctgg caactacttc aaggtttcga gagttaaaactccaacacaa 1260 agaatatctc tgtgtcaagg ccatgatcct gctcaattcc agtatgtaccctctggtcac 1320 agcgacccag gatgctgaca gcagccggaa gctggctcac ttgctgaacgccgtgaccga 1380 tgctttggtt tgggtgattg ccaagagcgg catctcctcc cagcagcaatccatgcgcct 1440 ggctaacctc ctgatgctcc tgtcccacgt caggcatgcg agtaacaagggcatggaaca 1500 tctgctcaac atgaagtgca aaaatgtggt cccagtgtat gacctgctgctggagatgct 1560 gaatgcccac gtgcttcgcg ggtgcaagtc ctccatcacg gggtccgagtgcagcccggc 1620 agaggacagt aaaagcaaag agggctccca gaacccacag tctcagtgacgcctggccct 1680 gaggtgaact ggcccacaga ggtcacaagc tgaagcgtga actccagtgtgtcaggagcc 1740 tgggcttcat ctttctgctg tgtggtccct catttggtga tggcaggcttggtcatgtac 1800 catccttccc tccaccttcc caactctcag gagtcggtgt gaggaagccatagtttccct 1860 tgttagcaga gggacatttg aatcgagcgt ttccacac 1898 25 530PRT Homo sapiens 25 Met Asp Ile Lys Asn Ser Pro Ser Ser Leu Asn Ser ProSer Ser Tyr 1 5 10 15 Asn Cys Ser Gln Ser Ile Leu Pro Leu Glu His GlySer Ile Tyr Ile 20 25 30 Pro Ser Ser Tyr Val Asp Ser His His Glu Tyr ProAla Met Thr Phe 35 40 45 Tyr Ser Pro Ala Val Met Asn Tyr Ser Ile Pro SerAsn Val Thr Asn 50 55 60 Leu Glu Gly Gly Pro Gly Arg Gln Thr Thr Ser ProAsn Val Leu Trp 65 70 75 80 Pro Thr Pro Gly His Leu Ser Pro Leu Val ValHis Arg Gln Leu Ser 85 90 95 His Leu Tyr Ala Glu Pro Gln Lys Ser Pro TrpCys Glu Ala Arg Ser 100 105 110 Leu Glu His Thr Leu Pro Val Asn Arg GluThr Leu Lys Arg Lys Val 115 120 125 Ser Gly Asn Arg Cys Ala Ser Pro ValThr Gly Pro Gly Ser Lys Arg 130 135 140 Asp Ala His Phe Cys Ala Val CysSer Asp Tyr Ala Ser Gly Tyr His 145 150 155 160 Tyr Gly Val Trp Ser CysGlu Gly Cys Lys Ala Phe Phe Lys Arg Ser 165 170 175 Ile Gln Gly His AsnAsp Tyr Ile Cys Pro Ala Thr Asn Gln Cys Thr 180 185 190 Ile Asp Lys AsnArg Arg Lys Ser Cys Gln Ala Cys Arg Leu Arg Lys 195 200 205 Cys Tyr GluVal Gly Met Val Lys Cys Gly Ser Arg Arg Glu Arg Cys 210 215 220 Gly TyrArg Leu Val Arg Arg Gln Arg Ser Ala Asp Glu Gln Leu His 225 230 235 240Cys Ala Gly Lys Ala Lys Arg Ser Gly Gly His Ala Pro Arg Val Arg 245 250255 Glu Leu Leu Leu Asp Ala Leu Ser Pro Glu Gln Leu Val Leu Thr Leu 260265 270 Leu Glu Ala Glu Pro Pro His Val Leu Ile Ser Arg Pro Ser Ala Pro275 280 285 Phe Thr Glu Ala Ser Met Met Met Ser Leu Thr Lys Leu Ala AspLys 290 295 300 Glu Leu Val His Met Ile Ser Trp Ala Lys Lys Ile Pro GlyPhe Val 305 310 315 320 Glu Leu Ser Leu Phe Asp Gln Val Arg Leu Leu GluSer Cys Trp Met 325 330 335 Glu Val Leu Met Met Gly Leu Met Trp Arg SerIle Asp His Pro Gly 340 345 350 Lys Leu Ile Phe Ala Pro Asp Leu Val LeuAsp Arg Asp Glu Gly Lys 355 360 365 Cys Val Glu Gly Ile Leu Glu Ile PheAsp Met Leu Leu Ala Thr Thr 370 375 380 Ser Arg Phe Arg Glu Leu Lys LeuGln His Lys Glu Tyr Leu Cys Val 385 390 395 400 Lys Ala Met Ile Leu LeuAsn Ser Ser Met Tyr Pro Leu Val Thr Ala 405 410 415 Thr Gln Asp Ala AspSer Ser Arg Lys Leu Ala His Leu Leu Asn Ala 420 425 430 Val Thr Asp AlaLeu Val Trp Val Ile Ala Lys Ser Gly Ile Ser Ser 435 440 445 Gln Gln GlnSer Met Arg Leu Ala Asn Leu Leu Met Leu Leu Ser His 450 455 460 Val ArgHis Ala Ser Asn Lys Gly Met Glu His Leu Leu Asn Met Lys 465 470 475 480Cys Lys Asn Val Val Pro Val Tyr Asp Leu Leu Leu Glu Met Leu Asn 485 490495 Ala His Val Leu Arg Gly Cys Lys Ser Ser Ile Thr Gly Ser Glu Cys 500505 510 Ser Pro Ala Glu Asp Ser Lys Ser Lys Glu Gly Ser Gln Asn Pro Gln515 520 525 Ser Gln 530 26 30 DNA Homo sapiens 26 gtgcggatcc tctcaagacatggatataaa 30 27 25 DNA Homo sapiens 27 agtaacaggg ctggcgcaac ggttc 2528 22 DNA Homo sapiens 28 actggcgatg gaccactaaa gg 22

What is claimed is:
 1. An isolated protein comprising an amino acidsequence selected from the group consisting of an amino acid sequence asset forth in SEQ. ID NO. 3, an amino acid sequence as set forth in SEQ.ID NO. 4, an amino acid sequence as set forth in SEQ. ID NO. 5, an aminoacid sequence as set forth in SEQ. ID NO. 6, an amino acid sequence asset forth in SEQ. ID NO. 21, and an amino acid sequence as set forth inSEQ. ID NO.
 25. 2. The protein according to claim 1, said proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:21 and SEQ ID NO:25.
 3. A chimericprotein having an N-terminal domain, a DNA-binding domain, and aligand-binding domain, wherein (a) the DNA-binding domain of thechimeric protein is selected from the group consisting of (1) aDNA-binding domain of estrogen receptor β which comprises an amino acidsequence as set forth in SEQ ID NO: 3, and (2) a DNA-binding domain of aprotein of the nuclear receptor superfamily and (b) the ligand-bindingdomain of the chimeric protein as selected from the group consisting of(1) a ligand-binding domain of estrogen receptor β which comprises anamino acid sequence as set forth in SEQ ID NO: 4 and (2) aligand-binding domain of a protein of the nuclear receptor superfamily,provided that the DNA-binding domain and the ligand-binding domain ofsaid chimeric protein are from different proteins, wherein the chimericprotein comprises either the DNA-binding domain of estrogen receptor βor the ligand-binding domain of estrogen receptor β.
 4. A proteinencoded by a recombinant expression vector comprising an isolated DNAencoding a protein having an N-terminal domain, DNA-binding domain, anda ligand-binding domain, wherein the amino acid sequence of saidDNA-binding domain of said protein comprises an amino acid sequence asset forth in SEQ ID NO: 3, and the amino acid sequence of saidligand-binding domain of said protein comprises an amino acid sequenceas set forth in SEQ ID NO:
 4. 5. The chimeric protein of claim 3,wherein the protein of the nuclear receptor superfamily is selected fromthe group consisting of a glucocorticoid receptor, a mineralcorticoidreceptor, a progestin receptor, an androgen receptor and an estrogenreceptor.